Team:Cambridge/HumanPractices/Interview

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To this end, we conducted an interview with Dr. Konrad Siegfried, a member of the team, and perhaps the most experienced user of the ARSOlux kit in the field. Our initial discussions focussed upon the storage and distribution of the bioreporters. We were interested to see that they had used freeze-dried e.coli as their chassis. We had not really considered this as an option during our [[Team:Cambridge/Project/DesignProcess|design process]], as we assumed that the shelf life of such cells at room temperature would be severely limited. Viable construct containing e.coli cells have been reawakened after three months at room temperature, and after six month when kept at four degrees centigrade, implying that our original assumptions were incorrect.
+
To this end, we conducted an interview with Dr. Konrad Siegfried, a member of the team, and perhaps the most experienced user of the ARSOlux kit in the field. Our initial discussions focussed upon the storage and distribution of the bioreporters. We were interested to see that they had used freeze-dried e.coli as their chassis. We had not really considered this as an option during our [[Team:Cambridge/Overview/DesignProcess|design process]], as we assumed that the shelf life of such cells at room temperature would be severely limited. Viable construct containing e.coli cells have been reawakened after three months at room temperature, and after six month when kept at four degrees centigrade, implying that our original assumptions were incorrect.
However, only a few freeze-drying facilities exist in Bangladesh, potentially limiting the amount of kits which can be produced locally. As discussed in a [http://www.buet.ac.bd/itn/pages/apsudocs/review_of_locally_available_kits.pdf  report by Kabir], production of arsenic measurement devices within the country should be a high priority. As any biological laboratory should have the equipment to produce bacillus spores, we maintain that bacillus should be used if possible. Nevertheless, freeze dried e.coli are a far more viable option than we had initially realized, and may be a useful alternative if the transition to bacillus fails.  
However, only a few freeze-drying facilities exist in Bangladesh, potentially limiting the amount of kits which can be produced locally. As discussed in a [http://www.buet.ac.bd/itn/pages/apsudocs/review_of_locally_available_kits.pdf  report by Kabir], production of arsenic measurement devices within the country should be a high priority. As any biological laboratory should have the equipment to produce bacillus spores, we maintain that bacillus should be used if possible. Nevertheless, freeze dried e.coli are a far more viable option than we had initially realized, and may be a useful alternative if the transition to bacillus fails.  

Latest revision as of 00:55, 27 October 2012

Parts for a reliable and field ready biosensing platform

Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

One minute tour! :)

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Contents

Judging Form

  • Please help the judges by filling out this form. Tell them what medal you think you deserve and why. Tell them which special prizes you should win. Help them find your best parts. Show them how you thought about the safety of your project. Helping the judges will help you too.
  • Team: Cambridge
  • Region: Europe
  • iGEM Year:2012
  • Track:Foundational Advance
  • Project Name:Parts for a reliable and field ready biosensing platform
  • Project Abstract: Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field.

    We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

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iGEM Medals for non-software teams

  • We believe our team deserves the following medal:
    • Bronze
    • Silver
    • √Gold

Because we met the following criteria (check all that apply and provide details where needed)

Requirements for a Bronze Medal

  • √Register the team, have a great summer, and plan to have fun at the Regional Jamboree.
  • √Successfully complete and submit this iGEM 2012 Judging form.
  • √Create and share a Description of the team's project using the iGEM wiki and the team's parts using the Registry of Standard Biological Parts.
  • √Plan to present a Poster and Talk at the iGEM Jamboree.
  • √Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts. Including:
    • √Primary nucleaic acid sequence
    • √Description of function
    • √Authorship
    • Safety notes, if relevant.
    • √Acknowedgment of sources and references
  • √Submit DNA for at least one new BioBrick Part or Device to the Registry.

Additional Requirements for a Silver Medal

  • √Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device.
  • √Enter this information and other documentation on the part's 'Main Page' section of the Registry
    Part Number(s): [http://partsregistry.org/Part:BBa_K911004 BBa_K911004]

Additional Requirements for a Gold Medal: (one OR more)

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iGEM Prizes

All teams are eligible for special prizes at the Jamborees. more... To help the judges, please indicate if you feel you should be evaluated for any of the following special prizes:

  • √Best Human Practice Advance
  • √Best Experimental Measurement
  • Best Model

Please explain briefly why you should receive any of these special prizes:

Best Human Practice Advance:

We feel that we deserve this prize for three reasons:

  1. We explored the impacts, *both positive and negative*, of synthetic biology as a solution to real world problems, through interviewing professionals working in a relevant field, namely the impact of arsenic water contamination in Bangladesh.
  2. We recognized existing problems with the way the current direction of synthetic. On going through the registry we found that most of the characterization data for biosensing parts is often neither comparable nor replicable. We have worked to solve this issue, for example with our ratiometric dual channel output.
  3. *Our project doesn’t stop here*, in Chanel number 6 (Team:Cambridge/HumanPractices/FutureDirections) we considered the future implications and technological applications of our project, as well as the means by which it could be improved by subsequent users. We feel that the end to an iGEM project should not be the conclusion of an idea, but the start of it.

Best BioBrick Measurement Approach:

It is absolutely vital that a quantitative, numerical, robust, and flexible measurement approach exists to relay information to a user that is an accurate representation of the input processed by a biological device. Working from these principles, the following was done:

  1. We designed and built Biologger, a *cheap, arduino-based, fully functional automatic rotary device* that has an incorporated ratiolumnometer
  2. Our project is entirely open-sourced and open-platform. We have published source code for the two applications which serve to operate the device, one for PCs and the other for Android devices, as well as the open source circuit design that provides this ratiometric reading. Furthermore, the Android app is able to receive its data wirelessly, which we feel is a great advance in BioBrick measurement.
  3. Our dual-channel luciferase reporter was successfully tested with a dilution series of E.coli transformed with the Lux Operon (under pBAD) biobrick (Part BBa_K325909) of the Cambridge iGEM 2010 team. It can detect, with good accuracy, both different light intensities, as well as the percentages of blue or orange frequencies in a sample.
  4. Our device was successfully tested using artificial light to detect different frequencies (colours) as well.

Having done all the above, we believe that this fully open-sourced instrumentation kit (mechanical) chassis, electronics, software code), estimated at *$35.00* (or $85.00 if a Bluetooth modem is required), is a complete BioBrick measurement solution for any and all BioBricks with a light output.

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Team_Parts

To help the judges evaluate your parts, please identify 3 of your parts that you feel are best documented and are of the highest quality.

  • Best new BioBrick part (natural)
    [http://partsregistry.org/Part:BBa_K911003 BBa_K911003]
    Best new BioBrick part (engineered)
    [http://partsregistry.org/Part:BBa_K911004 BBa_K911004]
  • Best improved part(s): None

List any other parts you would like the judges to examine:[http://partsregistry.org/Part:BBa_K911001 BBa_K911001], [http://partsregistry.org/Part:BBa_K911008 BBa_K911009], [http://partsregistry.org/Part:BBa_K911008 BBa_K911008]

Please explain briefly why the judges should examine these other parts:

  • Magnesium Sensitive Riboswitch [http://partsregistry.org/Part:BBa_K911001 BBa_K911001]
    As a riboswitch sensing construct, this part is an entirely new type of biosensor (along with the fluoride construct) that could potentially change the way we think about designing input genetic circuits. Unlike the fluoride riboswitch, it is a derepression system and therefore serves to demonstrate the principle that riboswitches can be used regardless of whether they turn on or off their reporter.
  • Fluorescent ratiometric construct for standardizing promoter output [http://partsregistry.org/Part:BBa_K911009 BBa_K911009]
    Fluorescence is a major cornerstone for biosensors in the registry, however, most parts do not involve the use of a ratiometric output, which has been shown in the literature to provide much more reliable and meaningful data. This part not only furthers the development of ratiometric measurements in molecular biology but due to the choice of promoters and terminators it can be used to characterize the difference in activity between E. coli and B. Subtilis
  • Fast Germination (B. subtilis) [http://partsregistry.org/Part:BBa_K911008 BBa_K911008]
    This part is entirely novel for the registry and fully utilizes the recombination machinery inherent in the Bacillus chassis. Have spores that can germinate at a faster rate is certainly a worthy achievement and could help with experiments with B. Subtilis that any future iGEM teams may wish to perform.

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iGEM Safety

For iGEM 2012 teams are asked to detail how they approached any issues of biological safety associated with their projects.

The iGEM judges expect that you have answered the four safety questions Safety page on your iGEM 2012 wiki.

Please provide the link to that page: Page name: Team:Cambridge/Safety

Attribution and Contributions

For iGEM 2012 the description of each project must clearly attribute work done by the team and distinguish it from work done by others, including the host labs, advisors, and instructors.

Please provide the link to that page, or comments in the box below: Page name: Team:Cambridge/Attributions

Comments

If there is any other information about your project you would like to highlight for the judges, please provide a link to your wiki page here: Team:Cambridge/Overview/DesignProcess

An Interview with Konrad Siegfried

We have aimed to create a system that would be directly usable in the field. Regarding the practical implementation of the system we have considered the challenges faced by end users.

A kit has been developed by the [http://www.ufz.de/arsolux/index.php?en=20675 ARSOlux team] which can be used for a real application, in this case the detection of arsenic contamination of ground water. They have recently tested their luminescence based kit in Bangladesh, showing some considerable improvements over traditional chemical solutions. Their experiences are documented in [http://pubs.acs.org/doi/abs/10.1021/es203511k this paper], in which they demonstrate that the use of bioreporters in such environments is both feasible and practical. We wanted more details about the system that they used, in order to understand how we could alter our design to fit within current policies and still be useful.

Discussions

KonradPic.jpg

To this end, we conducted an interview with Dr. Konrad Siegfried, a member of the team, and perhaps the most experienced user of the ARSOlux kit in the field. Our initial discussions focussed upon the storage and distribution of the bioreporters. We were interested to see that they had used freeze-dried e.coli as their chassis. We had not really considered this as an option during our design process, as we assumed that the shelf life of such cells at room temperature would be severely limited. Viable construct containing e.coli cells have been reawakened after three months at room temperature, and after six month when kept at four degrees centigrade, implying that our original assumptions were incorrect.

However, only a few freeze-drying facilities exist in Bangladesh, potentially limiting the amount of kits which can be produced locally. As discussed in a [http://www.buet.ac.bd/itn/pages/apsudocs/review_of_locally_available_kits.pdf report by Kabir], production of arsenic measurement devices within the country should be a high priority. As any biological laboratory should have the equipment to produce bacillus spores, we maintain that bacillus should be used if possible. Nevertheless, freeze dried e.coli are a far more viable option than we had initially realized, and may be a useful alternative if the transition to bacillus fails.

We were also interested in their apparent choice to use disinfection followed by autoclaving in a university laboratory as their biocontainment method. Upon questioning this form of bacterial destruction, it transpired that this was the only viable option that the Bangladeshi and German governments permit. Alternatively, the used equipment could have been sterilized after use with boiling water. Such biocontainment methods that we have proposed (the most promising simply being the release of bleach into the culture) would first have to be approved by similar authorities before they could be implemented, delaying application of our assay by years. Initial tests would therefore most likely be conducted using similar biocontainment methods to the ARSOlux team.

The kit developed by the ARSOlux team. Images from the ARSOlux website.

Despite this, Dr. Siegfried mentioned that his team were investigating self-destruction methods, which could be applied to considerably simplify the disposal of the bioreporter cells. One of the most promising avenues of research is apparently the application of chemicals within the vials that cause destruction of the cells upon exposure to sunlight, an incredibly simple measure to apply. Such simplicity also makes it more likely that these protocols will be followed, especially if similar tests are eventually released to the general public. Lastly, we wished to focus upon the policies enacted by the Bangladeshi government regarding the use of genetically modified bioreporters, as well as their stance upon the arsenic groundwater contamination problem. Particularly interesting to us is the fact that the governmental acceptable limit of arsenic is 50μg/l, while the WHO recommendation is 10μg/l. We asked Dr. Siegfried why he believed this was.

Graph showing the remarkable sensitivity and reliablitiy of the ARSOlux kit. Images from the ARSOlux website.

It seems that the Government of Bangladesh does not presently have the resources to tackle the vast arsenic contamination problem that faces its country. Without the means to deal with such a problem, it seems that they view the measurement of arsenic levels to within the WHO standard as moot, as a high proportion of the wells in the country will have higher arsenic levels. Additionally, the sensitivity of easily available chemical kits falls more within the 50μg/l range.

Sensing of arsenic levels is still important however, as in villages where the arsenic levels are low enough in some wells, well switching can be used as a tactic for reducing exposure to arsenic.

This led to a discussion about the application of the kit. Currently the test kit is not for sale in any country. At this point in time, it is used for scientific purposes and awareness-raising. The tests that have been performed by the ARSOlux team in Germany have been conducted in a specially fitted bus and with trained professionals operating the system. Their current plans are to use this bus to travel around different villages and to characterize the arsenic contamination present in the wells of an entire village in a single day. However, the water table of Bangladesh is in constant flux, Bangladesh being one of the most waterlogged countries in the world. The changing state of the aquifers in certain contaminated areas makes it likely that the arsenic concentrations of the wells will change from month to month. Lifting the restrictions on GMO use by the villagers themselves would therefore be very useful, as it would allow continual monitoring of each well.

Conclusions

As a result of this interview, we propose the following list of priorities that future teams and present day policy makers should consider:

1) Develop a reliable self-destruction mechanism, robust enough to convince governments of the impossibility of accidental GMO release, or at least that the consequences of such a release should be minimal.

2) Develop the infrastructure necessary to produce such kits in the country of interest and encourage the set up businesses that will manufacture the kits. Competition in the market should help alleviate the image problems that have been faced by biocontainment measures in the past, such as terminator genes.

3) Encourage NGOs working in the countries affected to try existing systems such as the ARSOlux kit, and emphasise the non-profit nature of such initial steps. Much of the poor image that has faced genetic modification up to the present is based on the assumption that large genetic modification companies are not working to create the most effective products, but to create the most profitable products as cheaply as possible. Emphasizing the open-source nature of the components used in such assemblies may also help convince people of their benefits.

4) With the above three points in place, we hope that some of the laws that currently restrict distribution of such kits will be lifted. At this point, with the infrastructure and corporate structures in place for the on-site production of bioreporter kits, the kits should be distributed to villages to permit continual assessment of water quality. Funding for such distribution may come from NGOs, or the government, or even directly from the settlements themselves if the kits are made cheap enough.

We hope that these priorities, when used in South Asia, should also help the successful application of our own kit.

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